Description of Microarray Controls

Universal Microarray Standard Set

The PFGRC has designed a universal microarray standard set (UMSS) for use with all current and future PFGRC microarrays. The probe set consists of Cy3 and Cy5 end labeled 40-mer probes that are complementary to a set of 500 Arabidopsis thaliana 70-mer targets on PFGRC DNA microarrays. The UMSS is supplied as a ready to use mix of the Cy3 and Cy5 labeled oligonucleotides. A small aliquot is spiked into Cy-dye labeled nucleic acid samples just prior to hybridization on PFGRC microarrays.

The UMSS produces a set of reference fluorescent signal intensities and ratios that are generated independent of the user-specific experimental samples. Since we intentionally targeted a heavy representation of Cy3 and Cy5 signals in the mid-range of detection, the Cy3 and Cy5 signal intensities and ratios generated by the UMSS should be highly reproducible. These signals serve as a reference point to judge the success of microarray experiments, and troubleshoot potential problems.

The 500 UMSS 70-mers have been spotted on all PFGRC 70-mer microarrays produced, thus the reference is compatible with 70-mer microarrays supplied by the PFGRC at any time. The 70-mer set was not included on version 1 microarrays (PCR product microarrays) for Streptococcus pneumoniae, Staphylococcus aureus, Salmonella typhimurium, and Test microarrays.

Control transcripts

We have provided a set of controls for our amplicon-based microarrays. Control spots are a set of ten amplicons designed to hybridize with the control transcripts (RNA) that we provide. The control transcripts can be mixed with your experimental RNA samples before labeling so that both are labeled simultaneously. The spots have been printed onto the Streptococus pneumoniae, Staphylococcus aureus, Salmonella typhimurim and our Test microarrays.

We have identified four of these transcripts that we favor for use in microarray experimentation. The four different cRNAs, transcribed in vitro from SP6 promoter using the MAXIscriptTM Kit (Ambion Inc., Austin, TX), are plant-specific and display no cross-reactivity to other control or experimental targets.

These transcripts are not printed on any of the oligonucleotide arrays. Control transcripts will be printed only on Streptococcus pneumoniae, Staphylococcus aureus, Salmonella typhimurium , and Test microarrays.